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How Are Antibodies Measured?

Antibodies are immune proteins called immunoglobulins that are produced in response to the detection of a foreign object or antigen, such as bacteria, viruses or cancer cells. There are five major types of immunoglobulins in the body: IgA, IgG, IgM, IgE and IgD. Levels for each type of antibody are measured to test and treat for cancer or other infectious diseases. There are several types of methods used to measure antibodies for researcher or medical needs.
  1. Enzyme-Linked Immunosorbent Assay

    • Enzyme-Linked Immunosorbent Assay (ELISA) is used to determine the amount of antibody or the amount of protein bound to antibody in a blood sample. The test uses the antigen/antibody interaction, where the antigen and antibody form complexes. The antigen or antibody in the sample are labeled with an enzyme. After the antigen and antibody form complexes they are able to be detected on an ELISA plate. The enzyme on the complex reacts with a substrate to form a colored product that can be counted on a plate counter in order to determine the amount of antibodies present in the sample.

    Western Blot

    • The Western Blot technique is used to determine both the molecular weight of antibodies and the relative amounts of antibodies present in a sample. Western blot techniques use gel electrophoresis to separate the antibodies. The antibodies are transferred to a nitrocellulose sheet and incubated with generic proteins. A solution containing enzymes is added to bind to specific antibodies. Substrate is added and the antibodies are incubated to convert the antibody/enzyme complexes into a colored product that can be viewed and photographed in order to determine the amount of antibodies present.

    Fluorescence Flow Cytometry

    • Fluorescence flow cytometry is a technique that is able to count antibodies through the measurement of the cellular and fluorescent properties of antibodies. A laser passes through the cells and the measurements are determined through changes in light scattering, light absorption and light emission, as it passes through detectors in the sample. The resulting light changes are transferred to a computer software program and manipulated into digital signals that are displayed in a chart in order to obtain specific cell counts for different types of cells, such as antibodies.

    Haemocytometer

    • Cell counts on antibodies can be performed using a haemocytometer. A blood sample solution is prepared into a Eppendorf tube using a pipette. Trypan blue solution is added to the solution to mark any dead cells and the mixture to added to the haemocytometer. The haemocytometer contains grid lines in which the cell counts are made using a hand tally while viewing the grid lines under 10X power of a compound microscope.


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